Replication is a well-conserved and essential process in all organisms, replication components are potential antibiotic targets and misregulation of replication can promote oncogenesis in multicellular organisms. This application examines bacterial replication initiation, which serves as a simplified model due to the presence of a single chromosome with a single replication origin, oriC, and a single replication initiation protein, DnaA. DnaA binds to oriC and directs the assembly of the replication machinery. Nucleotide binding by DnaA regulates DnaA activity, but dissecting this regulation has proven difficult due to the essentiality of DnaA, the autoregulation of DnaA and the failure of oriC-based plasmid replication models to faithfully reproduce chromosomal replication. Using the bacterium Bacillus subtilis as a model, this application will: 1) determine the role of DnaA's nucleotide binding in regulating DnaA-oriC interaction, 2) map the interactions between B. subtilis oriC and DnaA in vivo, and 3) dissect the regulation of DnaA by YabA and the [unreadable]-clamp. To accomplish these aims, I will use site-directed mutagenesis to generate DnaA constructs that are locked into the nucleotide-empty, DnaA-ATP or DnaA-ADP forms. I will use these constructs to probe the role of nucleotide binding/hydrolysis in regulating B. subtilis DnaA-oriC interaction and open complex formation. DnaA's interaction with the chromosomal origin of replication will be probed directly on the bacterial chromosome using chromatin immunoprecipitation and in vivo chemical footprinting. By using a B. subtilis strain that can replicate from either oriC or a heterologous, DnaA-independent origin of replication, oriN, I will be able to characterize the effect of otherwise lethal mutations in DnaA on oriC binding in vivo, and thereby define the role of nucleotide binding in supporting replication initiation in vivo. Last, I will dissect the RIDA-like mechanism proposed to exist in B. subtilis by characterizing the role YabA and the [unreadable]-clamp play in regulating B. subtilis DnaA's nucleotide binding/hydrolysis, DnaA-oriC interaction and DnaA-mediated replication initiation. PUBLIC HEALTH RELEVANCE: This application characterizes the events that lead to genome duplication in bacteria. Because the machinery that duplicates a cell's genetic material is a potential target for antibiotics, and because misregulation of genome duplication can promote cancer formation in higher organisms, this work will help to define antibiotic targets and refine our understanding of the molecular events that can promote cancer.